Mitochondria play a pivotal role in intracellular Ca(2+) signalling by taking up and releasing the ion upon specific conditions. In order to do so, mitochondria depend on a number of factors, such as the mitochondrial membrane potential and spatio-temporal constraints. Whereas most of the basic principles underlying mitochondrial Ca(2+) handling have been successfully deciphered over the last 50 years using assays based on in vitro preparations of mitochondria or cultured cells, we have only just started to understand the actual physiological relevance of these processes in the whole animal. Recent advancements in imaging and genetically encoded sensor technologies have allowed us to visualise mitochondrial Ca(2+) transients in live mice. These studies used either two-photon microscopy or bioluminescence imaging of cameleon or aequorin-GFP Ca(2+) sensors, respectively. Both methods revealed a consistent picture of Ca(2+) uptake into mitochondria under physiological conditions even during very short-lasting elevations of cytosolic Ca(2+) levels. The big future challenge is to understand the functional impact of such Ca(2+) signals on the physiology of the observed tissue as well as of the whole organism. To that end, the development of multiparametric in vivo approaches will be mandatory.